The peptide transport mechanism present in the small intestine and kidney plays an important role in protein nutrition. Protein digestion products are absorbed predominantly as small peptides via the intestinal peptide transporter. The renal peptide transporter functions to conserve the circulating peptides by providing a mechanism for their active reabsorption from the glomerular filtrate. The extraordinary ability to the intestine and kidney to absorb peptides forms the basis for the recent trend in clinical practice to substitute small peptides in the place of free amino acids in solutions used for enteral and parenteral nutrition. The peptide transporter is also currently receiving attention for its potential for therapeutic applications. The transport system acts as a vehicle for the transport of many pharmacologically active compounds, including the orally active peptide antibiotics such as aminocephalosporins. In addition to this nutritional, clinical and pharmacological significance, the peptide transporter is also biochemically unique due to its dependence on a proton motive force rather than a sodium motive force as the energy source. The goal of the current project is to study the molecular and regulatory aspects of this important transport system. The specific aims of the project include the cloning of the rabbit and human intestinal peptide transporters, structural and functional analyses of the cloned transporter and elucidation of the mechanisms involved in the hormonal regulation of the transporter activity. The proposed experimental approach to clone the transporter utilizes the Xenopus laevis oocyte expression system as a means to screen rabbit intestine and Caco-2 (a human intestinal cell line expressing the peptide transporter) cell cDNA libraries for the peptide transporter clone. In this approach, plasmid DNA will be transcribed in vitro to yield cRNA which will be 5'-capped and injected into oocytes, followed by measurement of H+-dependent dipeptide transport in injected clone. Antibodies will be generated against the cloned human intestinal engineered with the transporter cDNA. These antibodies will be utilized in the studies involving the regulation on the regulatory aspects of the transporter will be done in the current project using cultured cells of intestinal and renal origin. In these studies, the involvement of transcriptional and transnational processes, carrier recruitment and direct phosphorylation/dephosphorylation of the transporter protein in the regulation by hormones and second messengers will be investigated. Thus, the proposed experiment will lead to a clear understanding in two important areas in the field of peptide transport, molecular nature of the peptide transporter and hormonal regulation of this catalytic function.